title: Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R.
abstract: BACKGROUND
  Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU).
  
  
  METHODS
  Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation.
  
  
  RESULTS
  Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM.
  
  
  CONCLUSIONS
  We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.
authors.affiliation: Department of Oncology, Faculty of Medicine Dentistry and Health Sciences, The Institute for Cancer Studies, University of Sheffield, Sheffield S10 2RX, UK.
authors.forename: P
authors.initials: P
authors.lastname: Gravells
authors.forename: L
authors.initials: L
authors.lastname: Hoh
authors.forename: D
authors.initials: D
authors.lastname: Canovas
authors.forename: I G
authors.initials: IG
authors.lastname: Rennie
authors.forename: K
authors.initials: K
authors.lastname: Sisley
authors.forename: H E
authors.initials: HE
authors.lastname: Bryant
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000627:therapeutic use*; D004249:DNA Damage; D019008:Drug Resistance, Neoplasm; D005260:Female; D006657:Histones / Q000032:analysis; D006801:Humans; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology; D016685:Mitomycin / Q000627:therapeutic use*; D009251:NADPH-Ferrihemoprotein Reductase / Q000502:physiology*; D014604:Uveal Neoplasms / Q000188:drug therapy / Q000201:enzymology; D000098943:Uveal Melanoma
chemical_list: D000903:Antibiotics, Antineoplastic; D003432:Cross-Linking Reagents; C468783:H2AX protein, human; D006657:Histones; D016685:Mitomycin; D009251:NADPH-Ferrihemoprotein Reductase